Thymidine kinase

ABSTRACT

A polynucleotide comprising a nucleotide sequence encoding a thymidine kinase wherein at least one of the nucleotides corresponding to the splice donor site nucleotides is replaced by another nucleotide and wherein the nucleotides of the splice acceptor sites are not altered.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 14/676,696, filed Apr. 1,2015, which is a divisional of application Ser. No. 14/101,704, filedDec. 10, 2013, now U.S. Pat. No. 9,005,945, which is a divisional ofapplication Ser. No. 13/601,174, filed Aug. 31, 2012, now U.S. Pat. No.8,642,311, which is a continuation of application Ser. No. 11/629,962,filed Sep. 3, 2008, now U.S. Pat. No. 8,357,788, which is the NationalStage of International Application No. PCT/IB05/02358, filed on Jun. 17,2005, which claims priority under 35 U.S.C. § 119(a) to GB ApplicationNo. 0413702.2, filed Jun. 18, 2004, each of which is hereby incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 1, 2018, isnamed 048974_0001_04_583538_ST25, and is 17,756 bytes in size.

FIELD OF THE INVENTION

The present invention relates to a nucleic acid encoding a substantiallynon-splicing thymidine kinase gene and its use in therapy.

BACKGROUND TO THE INVENTION

There is considerable interest in the use of metabolic suicide genes ingene therapy. Numerous suicide genes are described in the literature,such as, for example, the genes coding for cytosine deaminase, purinenucleoside phosphorylase or a thymidine kinase. Among these genes, thegene coding for herpes simplex virus type 1 thymidine kinase (HSV-tk) isespecially advantageous from a therapeutic standpoint. The HSV-tk geneexpresses an enzyme, which when used in combination with the nucleosideanalogue ganciclovir, is capable of specifically eliminating dividingcells. Of particular importance is the presence of a propagated toxicityeffect (“bystander” effect) which is associated with the use ofHSV-tk/ganciclovir. This effect manifests itself in the destruction notonly of the cells which have incorporated the thymidine kinase (tk)gene, but also the neighbouring cells.

The mechanism of action of the HSV-tk/ganciclovir system, may beoutlined as follows: mammalian cells modified to express the TK enzymeimplement the first step of phosphorylation of ganciclovir to yieldganciclovir monophosphate. Subsequently, cellular kinases enable thisganciclovir monophosphate to be metabolised successively to diphosphateand then triphosphate. The ganciclovir triphosphate thus generated thenproduces toxic effects by becoming incorporated in the DNA, andpartially inhibits the cellular DNA polymerase alpha, thereby causingDNA synthesis to be stopped and hence leading to cell death (Moolten,1986; Mullen, 1994).

The HSV-tk/ganciclovir system can be used in a large number oftherapeutic applications and numerous clinical trials have beenimplemented in the last decade.

Methods of using the HSV-tk gene in gene therapy are disclosed in, forexample, WO90/07936, U.S. Pat. Nos. 5,837,510, 5,861,290, WO 98/04290,WO 97/37542 and U.S. Pat. No. 5,631,236.

One interesting application of the HSV-tk/ganciclovir system is in theprevention of graft-versus-host disease (GvHD), a condition that caninterfere with the outcome of allogeneic bone marrow transplantation,the treatment of choice for many hematological malignancies. GvHD occurswhen T-cells in the transplanted stem cell graft begin to attack therecipient's body. Removal of T-cells from the graft may prevent GvHD butalso favours disease recurrence and graft rejection. To counter theseeffects, allogeneic bone marrow transplant patients can be treated byintroducing donor T lymphocytes after a delay following the allogeneicbone marrow transplant. Transferring the HSV-tk gene to donor Tlymphocytes allows their eradication after ganciclovir administration incase of the emergence of GvHD. In one trial, patients received a T-celldepleted bone marrow transplantation together with increasing doses ofdonor lymphocytes transduced with the HSV-tk gene (Tiberghien et al.,2001). Circulating HSV-tk-expressing cells could be detected for morethan one year after engraftment in all patients. Six out of the twelvepatients developed GvHD and received ganciclovir, substantially reducingthe number of circulating modified cells (85% average decrease inabsolute number).

Mutants in the HSV-tk gene have been made which increase its biologicalactivity. Examples of such mutant HSV-tk genes are described in, forexample, Kokoris et al (1999), WO 95/30007, U.S. Pat. No. 5,877,010, WO99/19466 and WO 95/14102. However, a serious problem associated with thethymidine kinase/ganciclovir system is the emergence of ganciclovirresistance in HSV-tk transduced cells. This is of particular importance,since the relative proportion of cells which are resistant toganciclovir may rapidly increase through the course of treatment.

The presence of ganciclovir resistance in a lymphoblastoid human T-cellline transduced with a retroviral vector containing the HSV-tk gene wasfound to be associated with a 227 base pair deletion in the HSV-tk gene(Fillat et al., 2003). The same deletion was also present in humanprimary T cells transduced with the vector and in 12 patients whoreceived transduced donor T cells (Garin et at, 2001). WO 01/79502discloses that the cause of this deletion is believed to be due to thepresence of nucleotide sequences in the HSV-tk mRNA which act as splicesites to cause the production of a proportion of virus particlescarrying an aberrant form of the gene, the remainder carrying the fulllength gene. A mutant of the thymidine kinase gene is disclosed in WO01/79502 and in Chalmers 2001, Molecular Therapy 4:146-148, in which thesplice sites are removed, and which reduces the production of theaberrant form of the thymidine kinase gene. However, this mutant isstill associated with a detrimental amount of gene splicing. CD34-tkfusion constructs are disclosed in Rettig et al., 2003. These fusionconstructs contain modified HSV-tk genes. However, there is nodemonstration that the modified genes reduce splicing of the HSV-tkmRNA.

Thus, there remains a need for a modified thymidine kinase gene that isnot susceptible to gene splicing and which addresses the problemsassociated with ganciclovir resistance.

SUMMARY OF THE INVENTION

The present invention overcomes the aforementioned problem by providinga HSV-tk gene that expresses a greater proportion of unspliced mRNA overpreviously disclosed HSV-tk genes. We have identified a splice acceptorsite 14 base pairs upstream of the previously identified acceptor site.We show that previous attempts to inhibit splicing by modification ofthe previously identified donor and acceptor sites can result inactivation of this acceptor site and subsequent gene splicing. Wesurprisingly demonstrate that the modification of the HSV-tk gene atcertain specific nucleotides provides a HSV-tk gene that is capable ofexpressing substantially splice-free mRNA. In particular, we show thatmutating at least one of the nucleotides at the splice donor site cansubstantially prevent splicing.

STATEMENTS OF THE INVENTION

According to a first aspect of the present invention there is provided apolynucleotide comprising a nucleotide sequence encoding a thymidinekinase wherein at least one of the nucleotides corresponding to splicedonor site nucleotides is replaced by another nucleotide and wherein thenucleotides of the splice acceptor sites are not altered.

Preferably, at least one of the nucleotides corresponding to splicedonor site nucleotides at positions 329 and 330 of the wild typesequence in FIGS. 1A-1C (Tk wt) is replaced by another nucleotide andwherein the nucleotides of the splice acceptor sites are not altered.

In one embodiment, the nucleotide at position 330 is changed from T toC.

According to a second aspect of the present invention there is provideda polynucleotide comprising a nucleotide sequence encoding a thymidinekinase wherein at least one of the nucleotides corresponding to splicedonor site nucleotides at positions 329 and 330 of the wild typesequence in FIGS. 1A-1C (Tk wt) is replaced by another nucleotide andwherein the nucleotides of the splice acceptor sites are not altered.

In one embodiment both the nucleotides corresponding to splice donorsite nucleotides at positions 329 and 330 of the wild type sequence inFIGS. 1A-1C are replaced by another nucleotide.

In one embodiment the nucleotide at position 330 is changed from T to C.

According to a third aspect of the present invention there is provided apolynucleotide comprising a nucleotide sequence encoding a thymidinekinase wherein at least one of the nucleotides corresponding to thesplice donor site nucleotides at positions 329 and 330 of the wide typesequence in FIGS. 1A-1C (Tk wt) is replaced by another nucleotide andwherein at least one of the nucleotides corresponding to splice acceptorsite nucleotides at positions 554 and 555 and at least one of thenucleotides corresponding to splice acceptor site nucleotides atpositions 662 and 663 of the wild type sequence in FIGS. 1A-1C is notaltered.

Preferably both the nucleotides corresponding to positions 554 and 555are not altered.

Preferably both the nucleotides corresponding to positions 662 and 663are not altered.

In one embodiment both the nucleotides corresponding to splice donorsite nucleotides at positions 329 and 330 of the wild type sequence inFIGS. 1A-1C are replaced by another nucleotide.

In one embodiment the nucleotide at position 330 is changed from T to C.

According to a fourth aspect of the present invention there is provideda polynucleotide comprising the TkMut2 sequence in FIGS. 1A-1C.

According to a fifth aspect of the present invention there is provided apolynucleotide comprising a nucleotide sequence encoding a thymidinekinase wherein at least one of the splice acceptor nucleotidescorresponding to the nucleotide(s) at positions 329 and 330 of the wildtype sequence in FIGS. 1A-1C (Tk wt) is replaced by another nucleotideand wherein at least one of the nucleotides corresponding to the spliceacceptor site nucleotides at positions 541 and 542 of the wild typesequence in FIGS. 1A-1C is replaced by another nucleotide.

In one embodiment both the nucleotides corresponding to splice donorsite nucleotides at positions 329 and 330 of the wild type sequence inFIGS. 1A-1C are replaced by, another nucleotide.

In one embodiment the nucleotide corresponding to position 330 ischanged from T to C.

In another embodiment both the nucleotides corresponding to spliceacceptor site nucleotides at positions 541 and 542 of the wild typesequence in FIGS. 1A-1C are replaced by another nucleotide.

In another embodiment the nucleotide corresponding to the nucleotide atpositions 541 is changed from A to T.

In another embodiment the nucleotide corresponding to the nucleotide atposition 542 is changed from G to C.

In another embodiment at least one of the nucleotides corresponding tothe splice acceptor site nucleotides at position 554 and 555 of the wildtype sequence in FIGS. 1A-1C are replaced by another nucleotide suchthat there is no splice site present.

In one embodiment both the nucleotides corresponding to splice acceptorsite nucleotides at positions 541 and 542 of the wild type sequence inFIGS. 1A-1C are replaced by another nucleotide such that there is nosplice site present.

In another embodiment the nucleotide corresponding to the nucleotide atposition 555 is changed from G to A.

According to a sixth aspect of the present invention there is provided apolynucleotide comprising the TkMut23 sequence in FIGS. 1A-1C.

According to a seventh aspect of the present invention there is provideda polynucleotide comprising the TkMut234 sequence in FIGS. 1A-1C.

According to an eighth aspect of the present invention there is provideda polynucleotide comprising a nucleotide sequence encoding a thymidinekinase wherein at least one of the splice acceptor site nucleotidescorresponding to the nucleotide(s) at positions 329 and 330 of the wildtype sequence in FIGS. 1A-1C (Tk wt) is replaced by another nucleotideand wherein the nucleotides corresponding to the splice acceptor sitenucleotides at positions 554 and 555 of the wild type sequence in FIGS.1A-1C is replaced by another nucleotide.

In one embodiment both the nucleotides corresponding to splice donorsite nucleotides at positions 329 and 330 of the wild type sequence inFIGS. 1A-1C are replaced by another nucleotide.

In another embodiment both the nucleotides corresponding to spliceacceptor site nucleotides at positions 554 and 555 of the wild typesequence in FIGS. 1A-1C are replaced by another nucleotide.

Preferably the nucleotide corresponding to the nucleotide at position330 is changed from T to C.

Preferably the nucleotide corresponding to the nucleotide at position555 is changed from G to A.

According to a ninth aspect of the present invention there is provided apolynucleotide comprising the Tk-Mut24 sequence in FIGS. 1A-1C.

According to a tenth aspect of the present invention there is provided apolynucleotide comprising a nucleotide sequence encoding a thymidinekinase wherein at least one of the nucleotides corresponding to spliceacceptor site nucleotides at position 554 and 555 of the wild typesequence in FIGS. 1A-1C (Tk wt) is replaced by another nucleotide.

In one embodiment both the nucleotides corresponding to splice donorsite nucleotides at positions 554 and 555 of the wild type sequence inFIGS. 1A-1C are replaced by another nucleotide.

In one embodiment the nucleotide corresponding to the nucleotide atposition 555 is changed from G to A.

In one embodiment at least one of the splice acceptor site nucleotidescorresponding to the nucleotides at positions 541 and 542 of the wildtype sequence in FIGS. 1A-1C is also replaced by another nucleotide suchthat there is no splice site present.

In another embodiment both the nucleotides corresponding to spliceacceptor site nucleotides at positions 541 and 542 of the wild typesequence in FIGS. 1A-1C are also replaced by another nucleotide suchthat there is no splice site present.

In another embodiment the nucleotide corresponding to the nucleotide atposition 541 is changed from A to T.

In another embodiment the nucleotide corresponding to the nucleotide atposition 542 is changed from G to C.

According to an eleventh aspect of the present invention there isprovided a polynucleotide comprising the TkMut4 sequence in FIGS. 1A-1C.

According to a twelfth aspect of the present invention there is provideda polynucleotide comprising the TkMut34 sequence in FIGS. 1A-1C.

Preferably the replacement nucleotide does not alter the sequence of thepolypeptide encoded by said nucleotide sequence.

According to a thirteenth aspect of the present invention there isprovided a vector comprising a polynucleotide of the present invention.

Preferably, the vector is an expression vector.

According to a fourteenth aspect of the present invention there isprovided a host cell comprising a polynucleotide or a vector the presentinvention.

According to a fifteenth aspect of the present invention there isprovided a pharmaceutical composition comprising a polynucleotide, avector or a host cell of the present invention, and a pharmaceuticallyacceptable carrier.

According to a sixteenth aspect of the present invention there isprovided a kit comprising

-   -   (i) a polynucleotide, a vector, a host cell or a pharmaceutical        composition of the present invention; and    -   (i) a substantially non-toxic agent which is converted by        thymidine kinase into a toxic agent.

According to a seventeenth aspect of the present invention there isprovided a polynucleotide, a vector, a host cell or a pharmaceuticalcomposition of the present invention for use in medicine.

According to an eighteenth aspect of the present invention there isprovided products containing a polynucleotide, a vector, a host cell ora pharmaceutical composition of the present invention, and asubstantially non-toxic agent for simultaneous, separate or sequentialuse in treating a patient with cells in need of destruction, whereinsaid substantially non-toxic agent is converted by thymidine kinase to atoxic agent.

According to a nineteenth aspect of the present invention there isprovided a method of destroying cells comprising

-   -   (i) introducing into said cells a polynucleotide or a vector of        the present invention; and    -   (ii) simultaneously, separately or sequentially contacting said        cells with a substantially non-toxic agent which is converted by        thymidine kinase to a toxic agent.

According to a twentieth aspect of the present invention there isprovided a method of destroying cells comprising

-   -   (i) introducing into said cells a polynucleotide or a vector of        the present invention;    -   (ii) allowing said cells to express thymidine kinase; and    -   (ii) contacting said cells with a substantially non-toxic agent        which is converted by thymidine kinase to a toxic agent.

According to a twenty first aspect of the present invention there isprovided a method of treating a patient with cells in need ofdestruction comprising

-   -   (i) introducing into the patient a polynucleotide, a vector or a        pharmaceutical composition of the present invention;    -   (ii) simultaneously, separately or sequentially introducing into        the patient a substantially non-toxic agent which is converted        by thymidine kinase to a toxic agent.

According to a twenty second aspect of the present invention there isprovided a method of treating a patient with cells in need ofdestruction comprising

-   -   (i) introducing into the patient a polynucleotide, a vector or a        pharmaceutical composition of the present invention;    -   (ii) allowing said polynucleotide or vector to be taken up by        said cells;    -   (iii) allowing said cells to express thymidine kinase; and    -   (iv) introducing into the patient a substantially non-toxic        agent which is converted by thymidine kinase to a toxic agent.

According to a twenty third aspect of the present invention there isprovided a method of treating a patient with cells in need ofdestruction comprising

-   -   (i) removing the cells from the patient or donor cells;    -   (ii) introducing into the cells ex vivo a polynucleotide or a        vector of the present invention;    -   (iii) introducing the modified cells into the patient;    -   (iv) allowing the cells to express thymidine kinase; and    -   (v) administering to the patient a substantially non-toxic agent        which is converted by thymidine kinase into a toxic agent.

According to a twenty fourth aspect of the present invention there isprovided a method of preventing graft-versus-host disease in a patientcomprising:

-   -   (i) administering to a host T-cells genetically engineered to        include a polynucleotide of the present invention or a vector of        the present invention; and    -   (ii) administering to said host, prior to the occurrence of        graft-versus-host disease, a substantially non-toxic agent in an        amount effective to kill said genetically engineered T-cells        through interaction of said agent with thymidine kinase.

Preferably the substantially non-toxic agent used in the presentinvention is any one of ganciclovir, acyclovir, triflurothymidine,1-[2-deoxy, 2-fluoro, β-D-arabino furanosyl]-5-iodouracil, ara-A, ara 1,1-β-D arabino furanosyl thymine, 5-ethyl-2′deoxyurine,5-iodo-5′-amino-2, 5′-dideoxyuridine, idoxuridine, AZT, AIV,dideoxycytidine, Ara C and bromovinyl deoxyuridine (BVDU).

According to a twenty fifth aspect of the present invention there isprovided use of a polynucleotide, a vector or a host cell of the presentinvention in the preparation of a medicament for destroying cells in apatient.

According to a twenty sixth aspect of the present invention use ofpolynucleotide, vector or a host cell of the present invention in thepreparation of a medicament for the treatment of cancer.

DETAILED DESCRIPTION

Various preferred features and embodiments of the present invention willnow be described by way of non-limiting examples.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, 3. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D, McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press; andE. M. Shevach and W. Strober, 1992 and periodic supplements, CurrentProtocols in Immunology, John Wiley & Sons, New York, N.Y. Each of thesegeneral texts is herein incorporated by reference.

HSV-tk Gene

As used herein, the term “not altered” means not altered from the wildtype sequence (Tk wt).

As used herein, the term “replaced by another nucleotide” means replacedby a nucleotide that differs from the wild type sequence (Tk wt). Thereplacements are made such that the relevant splice donor site or spliceacceptor sites are removed.

Thymidine kinase mutants of the present invention may be prepared from awide variety of thymidine kinases. Preferably, the thymidine kinasemutant is derived from Herpesviridae thymidine kinase including forexample both primate herpesviruses, and nonprimate herpesviruses such asavian herpesviruses. Representative examples of suitable herpesvirusesinclude Herpes Simplex Virus Type 1 (McKnight et al., 1980), HerpesSimplex Virus Type 2 (Swain and Galloway, 1983), Varicella Zoster Virus(Davidson and Scott, 1986), marmoset herpesvirus (Otsuka and Kit, 1984),feline herpesvirus type 1 (Nunberg et al., 1989), pseudorabies virus(Kit and Kit, U.S. Pat. No. 4,514,497, 1985), equine herpesvirus type 1(Robertson and Whalley, 1988), bovine herpesvirus type 1 (Mittal andField, 1989), turkey herpesvirus (Martin et al., 1989), Marek's diseasevirus (Scott et al., 1989), herpesvirus saimiri (Honess et al., 1989)and Epstein-Barr virus (Baer et al., 1984).

Such herpesviruses may be readily obtained from commercial sources suchas the American Type Culture Collection (“ATCC”, Rockville, Md.).Deposits of certain of the above-identified herpesviruses may be readilyobtained from the ATCC, for example: ATCC No. VR-539 (Herpes Simplextype 1); ATCC Nos. VR-734 and VR-540 (Herpes Simplex type 2); ATCC No.VR-586 (Varicella Zoster Virus); ATCC No. VR-783 (Infectiouslaryngothracheitis); ATCC Nos. VR-624, VR-987, VR-2103, VR-2001,VR-2002, VR-2175, VR-585 (Marek's disease virus); ATCC Nos. VR-584B andVR-584B (turkey herpesvirus); ATCC Nos. VR-631 and VR-842 (bovineherpesvirus type 1); and ATCC Nos. VR-2003, VR-2229 and VR-700 (equineherpesvirus type 1). Herpesviruses may also be readily isolated andidentified from naturally occurring sources (e.g., from an infectedanimal).

The thymidine kinase gene may be readily isolated and mutated asdescribed below, in order to construct nucleic acid molecules encoding athymidine kinase gene comprising one or more mutations whichsubstantially reduce the splicing of the gene, as compared to unmutatedthymidine kinase. As utilized herein, it should be understood that“unmutated thymidine kinase” refers to native or wild-type thymidinekinase such as that described by McKnight et al. (Nucl. Acids Res.8:5949-5964, 1980).

It should be noted that in this application nucleotide positions arereferred to by reference to a position in FIGS. 1A-1C. However, whensuch references are made, it will be understood that the invention isnot to be limited to the exact sequence as set out in the figure butincludes variants and derivatives thereof. Thus, identification ofnucleotide locations in other thymidine kinase sequences arecontemplated (i.e., nucleotides at positions which the skilled personwould consider correspond to the positions identified in FIGS. 1A-1C).The person skilled in the art can readily align similar sequences andlocate the same nucleotide locations.

Construction of HSV-tk Mutants

Thymidine kinase mutants of the present invention may be constructedusing a variety of techniques. For example, mutations may be introducedat particular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes a derivative having the desired amino acid insertion,substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific (or segmentspecific) mutagenesis procedures may be employed to provide an alteredgene having particular codon altered according to the substitution,deletion, or insertion required. Deletion or truncation derivatives ofthymidine kinase mutants may also be constructed by utilizing convenientrestriction endonuclease sites adjacent to the desired deletion.Subsequent to restriction, overhangs may be filled in, and the DNAreligated. Exemplary methods of making the alterations set forth aboveare disclosed by Sambrook et al. (Molecular cloning: A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989).

Thymidine kinase mutants may also be constructed utilizing techniques ofPCR mutagenesis, chemical mutagenesis, chemical mutagenesis (Drinkwaterand Klinedinst, 1986) by forced nucleotide misincorporation (e.g., Liaoand Wise, 1990), or by use of randomly mutagenized oligonucleotides(Horwitz et al., 1989).

In a preferred embodiment of the present invention, the nucleotides aremodified taking note of the genetic code such that a codon is changed toa degenerate codon which codes fir the same amino acid residue. In thisway, it is possible to make coding regions of the protein of interestwhich encode wild type protein but which do not contain a functionalsplice site.

Splice Sites

The proportion of RNA which is removed (or “spliced out”) duringsplicing is typically called an intron, and the two pieces of RNA eitherside of the intron that are joined by splicing are typically calledexons (FIG. 2).

A splice donor site is a site in RNA which lies at the 5′ side of theRNA which is removed during the splicing process and which contains thesite which is cut and rejoined to a nucleotide residue within a spliceacceptor site. Thus, a splice donor site is the junction between the endof an exon and the start of the intron, typically terminating in thedinucleotide GU. In a preferred embodiment of the present invention, oneor both of the terminal GU dinucleotides (or GT dinucleotides in thecorresponding DNA sequence) of the splice donor site is/are altered toremove the splice site.

A splice acceptor site is a site in RNA which lies at the 3′ side of theRNA which is removed during the splicing process and which contains thesite which is cut and rejoined to a nucleotide residue within a splicedonor site. Thus, a splice acceptor site is the junction between the endof an intron (typically terminating with the dinucleotide AG) and thestart of the downstream exon. In a preferred embodiment of the presentinvention, one or both of the terminal AG dinucleotides of the spliceacceptor site is/are altered to remove the splice site.

Polynucleotides

Polynucleotides used in the invention may comprise DNA or RNA. They maybe single-stranded or double-stranded. It will be understood by askilled person that numerous different polynucleotides can encode thesame polypeptide as a result of the degeneracy of the genetic code. Inaddition, it is to be understood that skilled persons may, using routinetechniques, make nucleotide substitutions that do not affect thepolypeptide sequence encoded by the polynucleotides used in theinvention to reflect the codon usage of any particular host organism inwhich the polypeptides are to be expressed. The polynucleotides may bemodified by any method available in the art. Such modifications may becarried out in order to enhance the in vivo activity or life span ofpolynucleotides of the invention.

The polynucleotides used in the invention may encode fusion proteins forexample to aid in cellular secretion of the expressed polypeptides.Where, for example, the HSV-tk polypeptide is desired to be expressedfrom the cell, it may be desirable to add targeting sequences to targetthe proteins to particular cell compartments or to secretory pathways.Such targeting sequences have been extensively characterised in the art.

Polynucleotides such as DNA polynucleotides may be producedrecombinantly, synthetically, or by any means available to those ofskill in the art. They may also be cloned by standard techniques.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking a region of the lipid targeting sequencewhich it is desired to clone, bringing the primers into contact withmRNA or cDNA obtained from an animal or human cell, performing apolymerase chain reaction under conditions which bring aboutamplification of the desired region, isolating the amplified fragment(e.g. by purifying the reaction mixture on an agarose gel) andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan be cloned into a suitable cloning vector.

It will be appreciated that the polynucleotide of the invention maycontain only a coding region for the mutant thymidine kinase. However,it is preferred if the polynucleotide further comprises, in operablelinkage, a portion of nucleic acid that allows for efficient translationof the coding sequence in the target cell. It is further preferred ifthe polynucleotide (when in a DNA form) further comprises a promoter inoperable linkage which allows for the transcription of the coding regionand the portion of nucleic acid that allows for efficient translation ofthe coding region in the target cell.

Protein

As used herein, the term “protein” includes single-chain polypeptidemolecules as well as multiple-polypeptide complexes where individualconstituent polypeptides are linked by covalent or non-covalent means.As used herein, the terms “polypeptide” and “peptide” refer to a polymerin which the monomers are amino acids and are joined together throughpeptide or disulfide bonds. The terms subunit and domain may also referto polypeptides and peptides having biological function.

Variants, Derivatives, Analogues, Homologues and Fragments

In addition to the specific proteins and nucleotides mentioned herein,the present invention also encompasses the use of variants, derivatives,analogues, homologues and fragments thereof.

In the context of the present invention, a variant of any given sequenceis a sequence in which the specific sequence of residues (whether aminoacid or nucleic acid residues) has been modified in such a manner thatthe polypeptide or polynucleotide in question retains at least one ofits endogenous functions. A variant sequence can be obtained byaddition, deletion, substitution modification replacement and/orvariation of at least one residue present in the naturally-occurringprotein.

The term “derivative” as used herein, in relation to proteins orpolypeptides of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of and/oraddition of one (or more) amino acid residues from or to the sequenceproviding that the resultant protein or polypeptide retains at least oneof its endogenous functions.

The term “analogue” as used herein, in relation to polypeptides orpolynucleotides includes any mimetic, that is, a chemical compound thatpossesses at least one of the endogenous functions of the polypeptidesor polynucleotides which it mimics.

Typically, amino acid substitutions may be made, for example from 1, 2or 3 to 10 or 20 substitutions provided that the modified sequenceretains the required activity or ability. Amino acid substitutions mayinclude the use of non-naturally occurring analogues.

Proteins of use in the present invention may also have deletions,insertions or substitutions of amino acid residues which produce asilent change and result in a functionally equivalent protein.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe transport or modulation function is retained. For example,negatively charged amino acids include aspartic acid and glutamic acid;positively charged amino acids include lysine and arginine; and aminoacids with uncharged polar head groups having similar hydrophilicityvalues include leucine, isoleucine, valine, glycine, alanine,asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

“Fragments” are also variants and the term typically refers to aselected region of the polypeptide or polynucleotide that is of interesteither functionally or, for example, in an assay. “Fragment” thus refersto an amino acid or nucleic acid sequence that is a portion of afull-length polypeptide or polynucleotide.

Such variants may be prepared using standard recombinant DNA techniquessuch as site-directed mutagenesis. Where insertions are to be made,synthetic DNA encoding the insertion together with 5′ and 3′ flankingregions corresponding to the naturally-occurring sequence either side ofthe insertion site. The flanking regions will contain convenientrestriction sites corresponding to sites in the naturally-occurringsequence so that the sequence may be cut with the appropriate enzyme(s)and the synthetic DNA ligated into the cut. The DNA is then expressed inaccordance with the invention to make the encoded protein. These methodsare only illustrative of the numerous standard techniques known in theart for manipulation of DNA sequences and other known techniques mayalso be used.

Polynucleotide variants will preferably comprise codon optimisedsequences. Codon optimisation is known in the art as a method ofenhancing RNA stability and therefor gene expression. The redundancy ofthe genetic code means that several different codons may encode the sameamino-acid. For example, Leucine, Arginine and Serine are each encodedby six different codons. Different organisms show preferences in theiruse of the different codons. Viruses such as HIV, for instance, use alarge number of rare codons. By changing a nucleotide sequence such thatrare codons are replaced by the corresponding commonly used mammaliancodons, increased expression of the sequences in mammalian target cellscan be achieved. Codon usage tables are known in the art for mammaliancells, as well as for a variety of other organisms. Preferably, at leastpart of the sequence is codon optimised. Even more preferably, thesequence is codon optimised in its entirety.

Vectors

As it is well known in the art, a vector is a tool that allows orfacilitates the transfer of an entity from one environment to another.In accordance with the present invention, and by way of example, somevectors used in recombinant DNA techniques allow entities, such as asegment of DNA (such as a heterologous DNA segment, such as aheterologous cDNA segment), to be transferred into a host and/or atarget cell for the purpose of replicating the vectors comprising thenucleotide sequences used in the invention and/or expressing theproteins used in the invention. Examples of vectors used in recombinantDNA techniques include but are not limited to plasmids, chromosomes,artificial chromosomes or viruses.

Polynucleotides used in the invention are preferably incorporated into avector. Preferably, a polynucleotide in a vector for use in theinvention is operably linked to a control sequence that is capable ofproviding for the expression of the coding sequence by the host cell,i.e. the vector is an expression vector. The term “operably linked”means that the components described are in a relationship permittingthem to function in their intended manner. A regulatory sequence“operably linked” to a coding sequence is ligated in such a way thatexpression of the coding sequence is achieved under conditionscompatible with the control sequences.

The control sequences may be modified, for example by the addition offurther transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators.

Vectors may be transformed or transfected into a suitable host toprovide for expression of the tk gene product. This process may compriseculturing a host cell transformed with an expression vector as describedabove under conditions to provide for expression by the vector of acoding sequence encoding the protein, and optionally recovering theexpressed protein.

The vectors used in the present invention may be for example, plasmid orvirus vectors provided with an origin of replication, optionally apromoter for the expression of a polynucleotide and optionally aregulator of the promoter. The vectors may contain one or moreselectable marker genes, and/or a traceable marker such as GFP. Vectorsmay be used, for example, to transfect or transform a host cell.

Control sequences operably linked to sequences encoding proteins for usein the invention include promoters/enhancers and other expressionregulation signals. These control sequences may be selected to becompatible with the host cell for which the expression vector isdesigned to be used in. The term “promoter” is well-known in the art andencompasses nucleic acid regions ranging in size and complexity fromminimal promoters to promoters including upstream elements andenhancers.

The promoter is typically selected from promoters which are functionalin mammalian cells, although prokaryotic promoters and promotersfunctional in other eukaryotic cells may be used. The promoter istypically derived from promoter sequences of viral or eukaryotic genes.For example, it may be a promoter derived from the genome of a cell inwhich expression is to occur. With respect to eukaryotic promoters, theymay be promoters that function in a ubiquitous manner (such as promotersof a-actin, b-actin, tubulin) or, alternatively, a tissue-specificmanner (such as promoters of the genes for pyruvate kinase). Virolpromoters may also be used, for example the Moloney murine leukaemiavirus long terminal repeat (MMLV LTR) promoter, the rows sarcoma, virus(RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.

It may also be advantageous for the promoters to be inducible so thatthe levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated.

In addition, any of these promoters may be modified by the addition offurther regulatory sequences, for example enhancer sequences. Chimericpromoters may also be used comprising sequence elements from two or moredifferent promoters described above.

Preferably, the viral vector preferentially transduces a certain celltype or cell types.

Retroviral Vectors

In one embodiment, the vector used in the present invention is aretrovirus based vector which has been genetically engineered so that itcan not replicate and produce progeny infectious virus particles oncethe virus has entered the target cell. There are many retroviruses thatare widely used for delivery of genes both in tissue culture conditionsand in living organisms. Examples include and are not limited to murineleukemia virus (MLV), human immunodeficiency virus (HIV-1), equineinfectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV) and all other retroviridiae includinglentiviruses. A detailed list of retroviruses may be found in Coffin etat, 1997, “retroviruses”, Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763.

The basic structure of a retrovirus genome is a 5′ LTR and a 3′ LTR,between or within which are located a packaging signal to enable thegenome to be packaged, a primer binding site, integration sites toenable integration into a host cell genome and gag, pol and env genesencoding the packaging components—these are polypeptides required forthe assembly of viral particles. More complex retroviruses haveadditional features, such as rev and RRE sequences in HIV, which enablethe efficient export of RNA transcripts of the integrated provirus fromthe nucleus to the cytoplasm of an infected target cell.

In the provirus, these genes are flanked at both ends by regions calledlong terminal repeats (LTRs). The LTRs are responsible for proviralintegration, and transcription. LTRs also serve as enhancer-promotersequences and can control the expression of the viral genes.Encapsidation of the retroviral RNAs occurs by virtue of a psi sequencelocated at the 5′ end of the viral genome.

The LTRs themselves are identical sequences that can be divided intothree elements, which are called U3, R, and U5. U3 is derived from thesequence unique to the 3′ end of the RNA. R is derived from a sequencerepeated at both ends of the RNA and U5 is derived from the sequenceunique to the 5′ end of the RNA. The sizes of the three elements canvary considerably among different retroviruses.

In a defective retroviral vector genome gag, pol and env may be absentor not functional. The R regions at both ends of the RNA are repeatedsequences. U5 and U3 represent unique sequences at the 5′ and 3′ ends ofthe RNA genome respectively.

More preferably, the viral vector is a targeted vector, that is it has atissue tropism which is altered compared to the native virus, so thatthe vector is targeted to particular cells. This may be achieved bymodifying the retroviral Env protein. Preferably the envelope protein isa non-toxic envelope or an envelope which may be produced in non-toxicamounts within the primary target cell, such as for example a MMLVamphotropic envelope or a modified amphotropic envelope.

Preferably the envelope is one which allows transduction of human cells.Examples of suitable env genes include, but are not limited to, VSV-G, aMIX amphotropic env such as the 4070A env, the RD114 feline leukaemiavirus env or haemagglutinin (HA) from an influenza virus. The Envprotein may be one which is capable of binding to a receptor on alimited number of human cell types and may be an engineered envelopecontaining targeting moieties. The env and gag poi coding sequences aretranscribed from a promoter and optionally an enhancer active in thechosen packaging cell line and the transcription unit is terminated by apolyadenylation signal. For example, if the packaging cell is a humancell, a suitable promoter-enhancer combination is that from the humancytomegalovirus major immediate early (hCMV-MIE) gene and apolyadenylation signal from SV40 virus may be used. Other suitablepromoters and polyadenylation signals are known in the art.

MLV

Preferably, the retroviral vector used in the present invention is anMurine Leukemia Virus (MLV) vector. Retroviral vectors derived from theamphotropic Moloney murine leukemia virus (MLV-A) are commonly used inclinical protocols worldwide. These viruses use cell surface phosphatetransporter receptors for entry and then permanently integrate intoproliferating cell chromosomes. The genes are then maintained for thelifetime of the cell. Gene activity on MLV based constructs are easy tocontrol and can be effective over a long time. Clinical trials conductedwith these MLV-based systems have shown them to be well tolerated withno adverse side effects.

An example of an MLV vector for use in the present invention is a vectorderived from SFCMM-3, which carries both the suicide gene HSV-tk and themarker gene ΔLNGFR (Verzeletti 98, Human Gene Therapy 9:2243). Theoriginal vector used in the preparation of SFCMM-3 is LXSN (Miller etal. Improved retroviral vectors for gene transfer and expression.BioTechniques 7:980-990, 1989) (Genebank accession #28248). LXSN vectorwas modified by the insertion of the HSV-tk gone into the unique Hpa Isite (“blunt cut”), removal of the neo gene by digestion with Hind IIIand Nae I, and insertion of the cDNA encoding ΔLNGFR in this site.

Lentiviral Vector

In one embodiment, the vector of the present invention may be alentiviral vector. Lentivirus vectors are part of a larger group ofretroviral vectors. A detailed list of lentiviruses may be found inCoffin et al (“Retroviruses” 1997 Cold Spring Harbour Laboratory PressEds: J M Coffin, S M Hughes, H E Varmus pp 758-763). In brief,lentiviruses can be divided into primate and non-primate groups.Examples of primate lentiviruses include but are not limited to: thehuman immunodeficiency virus (HIV), the causative agent of humanacquired-immunodeficiency syndrome (AIDS), and the simianimmunodeficiency virus (SIV). The non-primate lentiviral group includesthe prototype “slow virus” visna/maedi virus (VMV), as well as therelated caprine arthritis-encephalitis virus (CAEV), equine infectiousanaemia virus (EIAV) and the more recently described felineimmunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells (Lewis, 1992; Lewis and Emerman, 1994).In contrast, other retroviruses—such as MLV—are unable to infectnon-dividing or slowly dividing cells such as those that make up, forexample, muscle, brain, lung and liver tissue. As lentiviruses are ableto transduce terminally differentiated/primary cells, the use of alentiviral screening strategy allows library selection in a primarytarget non-dividing or slowly dividing host cell.

Adenovirus Vectors

In another embodiment, the vector of the present invention may be anadenovirus vector. The adenovirus is a double-stranded, linear DNA virusthat does not go through an RNA intermediate. There are over 50different human serotypes of adenovirus divided into 6 subgroups basedon the genetic sequence homology. The natural target of adenovirus isthe respiratory and gastrointestinal epithelia, generally giving rise toonly mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) aremost commonly used in adenoviral vector systems and are normallyassociated with upper respiratory tact infections in the young.

Adenoviruses are nonenveloped, regular icosohedrons. A typicaladenovirus comprises a 140 nm encapsidated DNA virus. The icosahedralsymmetry of the virus is composed of 152 capsomeres: 240 hexons and 12pentons. The core of the particle contains the 36 kb linear duplex DNAwhich is covalently associated at the 5′ ends with the Terminal Protein(TP) which acts as a primer for DNA replication. The DNA has invertedterminal repeats (ITR) and the length of these varies with the serotype.

The adenovirus is a double stranded DNA nonenveloped virus that iscapable of in vivo and in vitro transduction of a broad range of celltypes of human and non-human origin. These cells include respiratoryairway epithelial cells, hepatocytes, muscle cells, cardiac myocytes,synoviocytes, primary mammary epithelial cells and post-mitoticallyterminally differentiated cells such as neurons.

Adenoviral vectors are also capable of transducing non dividing cells.This is very important for diseases, such as cystic fibrosis, in whichthe affected cells in the lung epithelium, have a slow turnover rate. Infact, several trials are underway utilising adenovirus-mediated transferof cystic fibrosis transporter (CFTR) into the lungs of afflicted adultcystic fibrosis patients.

Adenoviruses have been used as vectors for gene therapy and forexpression of heterologous genes. The large (36 kilobase) genome canaccommodate up to 8 kb of foreign insert DNA and is able to replicateefficiently in complementing cell lines to produce very high titres ofup to 10¹². Adenovirus is thus one of the best systems to study theexpression of genes in primary non-replicative cells.

The expression of viral or foreign genes from the adenovirus genome doesnot require a replicating cell. Adenoviral vectors enter cells byreceptor mediated endocytosis. Once inside the cell, adenovirus vectorsrarely integrate into the host chromosome.

Instead, it functions episomally (independently from the host genome) asa linear genome in the host nucleus. Hence the use of recombinantadenovirus alleviates the problems associated with random integrationinto the host genome.

Pox Virol Vectors

Pox viral vectors may be used in accordance with the present invention,as large fragments of DNA are easily cloned into their genome andrecombinant attenuated vaccinia, variants have been described (Meyer, etal., 1991; Smith and Moss, 1983).

Examples of pox viral vectors include but are not limited toleporipoxvirus: Upton, et at, 1986, (shope fibroma virus);capripoxvirus: Gershon, et al., 1989, (Kenya sheep-1); orthopoxvirus:Weir, et at, 1983, (vaccinia); Esposito, et al., 1984, (monkeypox andvariola virus); Hruby, et at, 1983, (vaccinia); Kilpatrick, et al.,1985, (Yaba monkey tumour virus); avipoxvirus: Binns, et al., (1988)(fowlpox); Boyle, et al., 1987, (fowlpox); Schnitzlein, et at, 1988,(fowlpox, quailpox); entornopox (Lytvyn, et al., 1992.

Poxvirus vectors are used extensively as expression vehicles for genesof interest in eukaryotic cells. Their ease of cloning and propagationin a variety of host cells has led, in particular, to the widespread useof poxvirus vectors for expression of foreign protein and as deliveryvehicles for vaccine antigens (Moss, 1991).

Vaccinia Virol Vectors

The vector of the present invention may be a vaccinia virus vector suchas MVA or NYVAC. Most preferred is the vaccinia strain modified virusankara (MVA) or a strain derived therefrom. Alternatives to vacciniavectors include avipox vectors such as fowlpox or canarypox known asALVAC and strains derived therefrom which can infect and expressrecombinant proteins in human cells but are unable to replicate.

Delivery Systems

The invention further provides a delivery system for mutant tkpolynucleotide of the present invention.

The delivery system of the present invention may be a viral or non-viraldelivery system. Non-viral delivery mechanisms include but are notlimited to lipid mediated transfection, liposomes, immunoliposomes,lipofectin, cationic, facial amphiphiles (CFAs) and combinationsthereof.

The polynucleotides may be delivered to the target cell population byany suitable Gene Delivery Vehicle, GDV. This includes but is notrestricted to, DNA, formulated in lipid or protein complexes oradministered as naked DNA via injection or biolistic delivery, virusessuch as retroviruses, adenoviruses, poxvirus, lentiviruses, herpesviruses, vaccinia viruses, adeno associated viruses, murine leukemiaviruses, semliki forest viruses and baculoviral viruses. Alternatively,the polynucleotides are delivered by cells such as monocytes,macrophages, lymphocytes or hematopoietic stem cells. In particular acell-dependent delivery system is used. In this system thepolynucleotide encoding the TK protein are introduced into one or morecells ex viva and then introduced into the patient.

The agents of the present invention may be administered alone but willgenerally be administered as a pharmaceutical composition.

Host Cells

Vectors and polynucleotides of the invention may be introduced into hostcells for the purpose of replicating the vectors/polynucleotides and/orexpressing the TK encoded by the polynucleotide of the invention. Thehost cell may be a bacterial cell or a eukaryotic cell, for example ayeast, insect or a mammalian cell.

The host cell may be a cell for packaging and propagating a virus, suchas retroviral packaging cell lines which are well known in the art.

The host cell may be a cell in an animal or patient (whether human oranimal) which it is desired to destroy. The polynucleotide and vector ofthe present invention are useful to target to cells to be destroyed.Cells expressing TK may be contacted with an agent which issubstantially non-toxic which is converted to a toxic form by TK.

Method of Destroying Cells

In one aspect of the present invention there is provided a method ofdestroying cells comprising

-   -   (i) introducing into said cells a polynucleotide or a vector of        the present invention;    -   (ii) allowing said cells to express thymidine kinase; and        contacting said cells with a substantially non-toxic agent which        is converted by thymidine kinase to a toxic agent.

The introduction into the cells of the polynucleotide or vector, and thecontacting of the cells with the substantially non-toxic agent, may bein any order. The cells to be destroyed may be in vitro, such as cellswhich are grown in culture, or they may be cells which are part of ananimal. Representative examples of cells which it is desired to destroyare T-cells, autoimmune cells, tumor cells, cells which do not expressor inappropriately express a particular gene, and cells infected withbacteria, viruses, or other intracellular parasites.

Treatment

It is to be appreciated that all references herein to treatment includecurative, palliative and prophylactic treatment. The treatment ofmammals is particularly preferred. Both human and veterinary treatmentsare within the scope of the present invention.

In one aspect of the present invention there is provided a method oftreating a patient with cells in need of destruction comprising

-   -   (i) introducing into the patient a polynucleotide, a vector or a        pharmaceutical composition of the present invention;    -   (ii) allowing said polynucleotide or expression vector to be        taken up by said cells;    -   (iii) allowing said cells to express thymidine kinase; and    -   (iv) introducing into the patient a substantially non-toxic        agent which is converted by thymidine kinase to a toxic agent.

In another aspect of the present invention there is provided a method oftreating a patient with cells in need of destruction comprising

-   -   (i) removing the cells from the patient or donor cells;    -   (ii) introducing into the cells ex vivo a polynucleotide or an        expression vector of the present invention;    -   (iii) introducing the modified cells into the patient;    -   (iv) allowing the cells to express thymidine kinase; and        administering to the patient a substantially non-toxic agent        which is converted by thymidine kinase into a toxic agent.

Representative examples of diseases which can be treated bypolynucleotides, vectors and methods of the present invention includediseases such as cancer, hyperkeratosis, prostate hypertrophy,hyperthyroidism, a wide variety of endocrinopathies, autoimmunediseases, allergies, restenosis, a wide variety of viral diseases suchas HIV and AIDS, hepatitis and intracellular parasitic diseases.

The polynucleotides, vectors and methods of the present invention may beused in the course of therapy following allogeneic bone marrowtransplant. Allogeneic bone marrow transplant is the treatment of choicefor many hematologic malignancies, such as leukemia, lymphoma andmultiple myeloma. Transplantation of allogeneic bone marrow,particularly when employed with high dose chemoradiotherapy, has beenshown to produce superior results compared to autologous or syngeneictransplants.

In performing allogeneic bone marrow transplant alloreactive Tlymphocytes may be removed from the graft to prevent graft versus hostdisease (GvHD). GvHD occurs when T-cells in the transplanted stem cellgraft may begin to attack the recipient's body. However, such removal ofcells can increases the incidence of disease relapse, graft rejectionand reactivation of viral infection. To counter these effects,allogeneic bone marrow transplant patients can be treated by introducingdonor T lymphocytes after a delay following the allogeneic bone marrowtransplant. The therapeutic promise of delayed introduction of donor Tlymphocytes following allogeneic bone marrow transplant, however, islimited by GvHD, a frequent and potentially lethal complication of thetreatment. This problem has been addressed by the administering to apatient T-cells genetically engineered to include a polynucleotideencoding a “suicide gene”. The polynucleotides and vectors of thepresent invention encoding the mutant TK may be been used in thisregard.

Thus, in one aspect of the present invention there is provided a methodof preventing graft-versus-host disease in a patient comprising

-   -   (i) administering to a host T-cells genetically engineered to        include a polynucleotide or vector of the present invention; and    -   (ii) administering to said host, prior to the occurrence of        graft-versus-host disease, a substantially non-toxic agent in an        amount effective to kill genetically engineered T-cells capable        of providing a graft-versus-host effect in said patient through        interaction of said agent with thymidine kinase.

The cells comprising the polynucleotide or vector encoding the mutant tkgene preferably comprises a marker gene which may be used to monitor thepresence of the mutant tk gene. Preferably, the marker gene is encodedby the polynucleotide or expression vector of the present invention. Anexample of one such marker gene is a modified low affinity nerve growthfactor receptor (ΔLNGFR). Modified LNGFR, when expressed on the surfaceof transduced cells retains the binding properties of the correspondingunmodified NGF receptor with respect to its ligand, yet cannot effectsignal transduction as a result of ligand binding. Examples of specificLNGFR modifications are described in U.S. application Ser. No.08/602,791.

Pharmaceutical Compositions

A pharmaceutical composition is a composition that comprises or consistsof a therapeutically effective amount of a pharmaceutically activeagent. It preferably includes a pharmaceutically acceptable carrier,diluent or excipients (including combinations thereof). Acceptablecarriers or diluents for therapeutic use are well known in thepharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Examples of pharmaceutically acceptable carriers include, for example,water, salt solutions, alcohol, silicone, waxes, petroleum jelly,vegetable oils, polyethylene glycols, propylene glycol, liposomes,sugars, gelatin, lactose, amylose, magnesium stearate, tale,surfactants, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, petroethral fatty acid esters,hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

Where appropriate, the pharmaceutical compositions can be administeredby any one or more of: inhalation, in the form of a suppository orpessary, topically in the form of a lotion, solution, cream, ointment ordusting powder, by use of a skin patch, orally in the form of tabletscontaining excipients such as starch or lactose, or in capsules orovules either alone or in admixture with excipients, or in the form ofelixirs, solutions or suspensions containing flavouring or colouringagents, or they can be injected parenterally, for exampleintracavernosally, intravenously, intramuscularly or subcutaneously. Forparenteral administration, the compositions may be best used in in theform of a sterile aqueous solution which may contain other substances,for example enough salts or monosaccharides to make the solutionisotonic with blood. For buccal or sublingual administration thecompositions may be administered in the form of tablets or lozengeswhich can be formulated in a conventional manner.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to be deliveredusing a mini-pump or by a mucosal route, for example, as a nasal sprayor aerosol for inhalation or ingestable solution, or parenterally inwhich the composition is formulated by an injectable form, for delivery,by, for example, an intravenous, intramuscular or subcutaneous route.Alternatively, the formulation may be designed to be delivered by bothroutes.

Further preferred features and embodiments of the present invention willnow be described by way of non-limiting example and with reference tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C, show the multiple alignment of nucleotidesequences of HSV-1 thymidine kinase gene (HSV-tk) mutants (TkMut2 (SEQID NO 2), TkMut23 (SEQ ID NO:3), TkMut24 (SEQ ID NO:4), TkMut34 (SEQ IDNO:5), TkMut4 (SEQ ID NO:6) and TkMut234 (SEQ ID NO: 7). Bases arenumbered from the ATG start codon of wild-type HSV-tk sequence.Sequences are aligned against the TK wt sequence (TK wt, SEQ ID NO:1).The position of mutations is indicated by a number above the alignment.Mutations at positions (330), (541 and 542), and (555) are representedby the numbers 2, 3 and 4 respectively.

FIG. 2 shows the splicing consensus sequence.

FIG. 3 shows the HSV-tk PCR product of cDNA prepared from thesupernatant of echotropic packaging cell line GP+E86 and from theamphotropic packaging cell line gp+env Am12 (Am12) transfected withSFCMM-3 (Verzelletti, 1998, Human Gene Therapy 9:2243-2251) and/or TK3(scSFCMM-3) (Chalmers, 2001) vector.

FIG. 4 shows PCR amplified HSV-tk in peripheral blood lymphocytes (PBL)transduced with SFCMM-3 and/or TK3 (scSFCMM-3) vector.

FIG. 5 shows the sequence of the critical portion of the HSV-tk gene inSFCMM-3 (SEQ ID NO: 8) versus TK3 (scSFCMM-3) (SEQ ID NO: 9). Thenucleotide positions different between SFCMM-3 and TK3 (scSFCMM-3)vectors are bordered by a square. The gene segments which are deleted inthe spliced forms of SFCMM-3 and TK3 are indicated in italics. Thecorresponding splicing donor and acceptor sites are indicated.

FIG. 6 shows a schematic summary of the mutations introduced into theSFCMM-3 vector. The depicted nucleotide sequence of the donor site isset forth in SEQ ID NO: 10. The depicted DNA sequence of the acceptorsite is set forth in SEQ ID NO: 12. The depicted amino acid sequence ofthe donor site is set forth in SEQ ID NO: 11. The depicted amino acidsequence of the acceptor site is set forth in SEQ ID NO: 13.

FIG. 7 shows HSV-tk PCR product of cDNA derived from supernatants of E86cells transiently transfected with recombinant plasmids derived fromSFCMM-3 comprising the HSV-tk mutants shown in FIGS. 1A-1C.

FIG. 8 shows HSV-tk. PCR product of cDNA derived from supernatants ofAm12 cultures which has been transduced with the supernatants collectedfrom the E86 cultures described in FIG. 7.

FIG. 9 shows TKA1/TKB1 HSV-tk PCR product of DNA derived from CEM A301cells transduced with the supernatants collected from the Am12 culturesdescribed in FIG. 8.

FIG. 10 shows TKY1/TKY2 and TKZ1/TKZ2 HSV-tk PCR products of DNA derivedfrom CEM A301 cells transduced with the supernatants collected from theAm12 cultures described in FIG. 8.

FIG. 11 shows TKA1/TKB1 HSV-tk PCR product of DNA derived from PBLstransduced with the supernatants collected from the Am12 culturesdescribed in FIG. 8.

FIG. 12 shows TKZ1/TKZ2 HSV-tk PCR product of DNA derived from PBLstransduced with the supernatants collected from the Am12 culturesdescribed in FIG. 8.

EXAMPLE 1—RELATIVE FREQUENCY OF THE SPLICED FORM OF HSV-TK IN SFCMM-3#35SUPERNATANT AND TRANSDUCED CELLS

The relative frequency of the spliced form (percent spliced form versusspliced plus unspliced) was evaluated by two quantitative real-timeRT-PCR (RNA from supernatant) or PCR (DNA from transduced lymphocytes),specific for each HSV-tk form, using TaqMan 7700 system.

Materials and Methods

SFCMM-3 Retroviral Vector

The GP+envAm12 (Am12)-derived producer cell line, SFCMM-3 clone 35(Am12/SFCMM-3#35), was expanded 5 days in DMEM (Cambrex) mediumcontaining 2 mM glutamine and 10% of irradiated FBS (Hyclone).Retroviral supernatants were harvested from confluent flasks in X-VIVO15 medium (Cambrex) supplemented with 2 mM glutamine after 24 hincubation of the cells at 33° C. Virus-containing supernatants werefiltered and stored at −80° C. until further use.

T Cell Transduction

Peripheral blood mononuclear cells were collected from healthy donorsand isolated by centrifugation on Lymphoprep (Nycomed).

Peripheral blood lymphocytes (PBLs) were stimulated with OKT3 (30 ng/ml)(Orthoclone) and cultured in RPMI (Hyclone) containing 3% of autologusplasma, 2 mM glutamine and supplemented with recombinant humaninterleukin 2, 600 IU/ml (Proleukin). Transduction of PBLs was performedby centrifugation of retroviral vector particles (supernatant fromAm12/SFCMM-3#35) on OKT3-stimulated PBLs.

Two centrifugation cycles were performed on the second and the third dayafter OKT3 stimulation.

After the transduction phase, lymphocytes were collected and thepercentage of actually transduced cells evaluated by FACS analysis,thanks to the ΔLNGFR molecule, used as a cell surface marker. Thetransduced cells were then sorted by antibodies conjugated to magneticbeads (Dynabeads), expanded a few days in culture, and then frozen aftera total of 10 days in culture.

Samples Preparation

DNA was purified from 1-2×10⁶ cells by using QIAamp DNA Mini kit(Qiagen) following manufacturer's protocols and yield was determined bymeasuring the OD₂₆₀. RNA was purified from 140 μl of supernatant byusing QIAamp Virol RNA Mini. Kit (Qiagen) according to themanufacturer's protocols. 13.5 μl of purified RNA were first subjectedto DNase treatment at 25° C. for 20 min, and 95° C. for 5 min toinactivate DNase. Half of the DNase-treated RNA was then reversetranscribed at 42° C. for 1 h using M-MLV Reverse Transcriptase(Invitrogen) and Oligo dT. Synthesized cDNA was 5-fold diluted beforeusing in the Real time PCR.

Quantitation of Spliced Form by Real Time PCR

DNA standard curve has been prepared by subcloning the unspliced andtruncated form of HSV-tk into pCR2.1 TOPO vector (Invitrogen). Twodifferent sets of primers and probes have been designed using PrimerExpress™ 1.5 software (PE Applied Biosystems), able to selectivelyamplify and detect the unspliced and the spliced form of HSV-tk. Twoquantitative real time PCRs were set-up, specific for each HSV-tk form,using TaqMan/ABI PRISM 7700 sequence detection system.

To set up a Quantitative Real time PCR specific for the HSV-tk unsplicedform, primers and probe were designed in the spliced region of HSV-tkgene. Real Time PCR for the unspliced form was performed in a 25 μlreaction mixture containing 100-500 ng of genomic DNA or 10 μl of cDNAprepared as described above, 1× TaqMan Universal PCR Master Mix (PEApplied Biosystems) 300 nM of each of the two primers TKwt for (5′-CGGCGG TGG TAA TGA CAA G-3′; SEQ ID NO: 14) and Tkwtrev (5′-GCG TCG GTC ACGGCA TA-3′; SEQ ID NO: 15) and 200 nM of TKwt MOB probe (5′-FAM CCA GATAAC AAT GGG C-3′; SEQ ID NO: 16).

A TaqMan probe encompassing the splice junction was designed toselectively detect the HSV-tk spliced form. Quantitative Real time PCRspecific for the TK spliced (truncated) form was performed in a 25 μlreaction mixture containing 100-500 ng of genomic DNA or 10 μl of cDNAprepared as described above, 1× Master Mix (PE Applied Biosystems) 300nM of each of the two primers TKSP18 (5′-GGA TGA GGG CCA CGA A-3′; SEQID NO: 17) and TKSP16 (5′-CGA ACA TCT ACA CCA CAC AAC A-3′; SEQ ID NO:18) and 200 nM of Taq Man probe PSP10 (5′ FAM-CCA GCA COG CCC TOGTCG-TAMRA 3′; SEQ ID NO: 19). Thermal cycling conditions were asfollows: initial activation of UNG at 50° C. for 2 min, followed byactivation of Taq Gold and inactivation of UNG at 95° C. for 15 min.Subsequently, 40 cycles of amplification were performed at 95° C. for 15s and 60° C. for 1 min. Both PCRs were performed in parallel in MicroAmpoptical 96-well reaction plates (Applied Biosystems) using the ABI Prism7700 Sequence Detection Systems (Applied Biosystems). Mean baselinefluorescence was calculated from PCR cycles 3 to 15, and Ct was definedas the PCR cycle in which the normalized fluorescence intensity of thereporter dye equaled 0.05. Two standard curves with known copy numbers(from 10⁶ to 4 copies/reaction) were generated in each TaqMan assay byplotting the Ct values against the logarithm of the initial input of DNAamount. Standard dilutions and cDNA samples were analyzed in duplicateand triplicate, respectively. Both PCRs were validated and showedextended dynamic range (6 log), high sensitivity (<10 copies/reaction)good reproducibility (CV<5%) and repeatability (CV<5%).

Results

The results are shown in Table 1. Six clinical grade lots of SFCMM-3vector supernatant were analysed, as well as nine preparations oftransduced lymphocytes.

The relative frequency of the spliced form is below 5% in all 6 clinicalgrade lots of SFCMM-3 supernatant (1.12+/−0.75, range 0.65-2.60), aswell as in 9 preparations of T lymphocytes transduced with SFCMM-3supernatant (1.89+/−1.22, range 0.84-4.00).

EXAMPLE 2—VARIANT OF SPLICED TK INS SCSFCMM-3 SUPERNATANTS ANDTRANSDUCED CELLS

Materials and Methods

scSFCMM-3 Retroviral Vector and Producer Cell Lines

Vector DNA TK3 (TK3 Molmed and TK3 are different preparations of thesame plasmid scSFCMM-3, described in: Chalmers 2001, Molecular Therapy4:146-148) was transfected into the echotropic packaging cell line GP+E-86 (E86) by calcium phosphate coprecipitation. The supernatantobtained from the transient transfection of E86 cells was filtered andthen used to infect the Am12 cell line. The fraction of cells containingTK3 was isolated by using immunomagnetic selection and the resultingAm12/TK3 bulk culture was expanded. After limiting dilution of theAm12/TK3 bulk culture, clones #53, #71, and #80 were selected on thebasis of high growth capacity and transduction efficiency on Tlymphocytes.

The Am12/TK3 clones were expanded in DMEM (Cambrex) containing 2 mMglutamine and 10% irradiated FBS (Hyclone). Retroviral supernatants wereharvested in X-VIVO 15 medium (Cambrex) supplemented with 2 mM glutamineafter 24 h incubation of the cells at 33° C. Virus-containingsupernatants were filtered with 0.22 μm filters and stored at −80° C.until further use.

T Cell Transduction

Peripheral blood mononuclear cells were collected from several healthydonors and isolated by centrifugation on Lymphoprep (Nycomed).

Peripheral blood lymphocytes (PBLs) were stimulated with OKT3 (30 ng/ml)(Orthoclone) and cultured in RPMI (Hyclone) containing 3% of autologusplasma, 2 mM glutamine and supplemented with recombinant humaninterleukin 2, 600 IU/ml (Proleukin). Transduction of PBLs was performedby centrifugation of retroviral vector particles on OKT3-stimulatedPBLs. Two centrifugation cycles were performed on the second and thethird day after OKT3 stimulation.

After the transduction phase, PBLs were collected and the percentage ofactually transduced cells evaluated by FACS analysis, thanks to theΔLNGFR molecule, used as a cell surface marker. The transduced cellswere then sorted by antibodies conjugated to magnetic beads (Dynabeads),expanded a few days in culture, and pellets were prepared for PCRanalysis.

Polymerase Chain Reaction

The bulk producer cell line (Am12/TK3 Bulk 2) as well as single producercell clones obtained by limiting dilution (Am12/TK3 #53, #71 and #80)were analysed. RNA was extracted from the culture supernatantscontaining infectious viral particles.

RT-PCR was performed with HTK4+ (5′-TTC TCT AGG CGC CCG AAT TCG TT-3′;SEQ ID NO: 20) and HTK2-(5′-ATC CAG GAT AAA GAC GTG CAT GG-3′; SEQ IDNO: 21) primers or TKA1 (5′-COT ACC CGA GCC GAT GAC TT-3′; SEQ ID NO:22) and TKB1 (5′-TGT GTC TOT CCT CCG GAA GG-3′; SEQ ID NO: 23) primers.RT PCR was performed using Titan One tube RT-PCR System (Roche) in a 50μl reaction mixture containing 10 μl of RNA DNase-treated, 1×RT-PCRBuffer with 1.5 mM of MgCl₂ (PE Applied Biosystems), 200 μM of eachdeoxynucleotide (dNTP), 200 nM of each primer, 5 mM of dithiothreitol(DTT), 20 U of RNase Inhibitor, 1 μl of Titan Enzyme mix.

Supernatants from Am12/SFCMM-3 #35 and DNA from SFCMM-3 transducedlymphocytes were used as controls. RT-controls were also run inparallel.

The RT-PCR cycling profile consisted of a first reverse transcriptionstep for 30 min at 50° C., a denaturation step for 2 min at 94° C.followed by 40 cycles with denaturation for 30 s at 95° C., annealingfor 30 s at 60° C. and elongation for 1 min 30 s at 68° C., and onefinal elongation step of 10 min at 68° C. Ten microliters of amplifiedproduct were analyzed by agarose gel electrophoresis.

Genomic DNA was extracted from PBLs of several donors transduced withSFCMM-3 or TK3 vector. PCR was performed in a 25 μl reaction mixturecontaining 100-500 ng of genomic DNA, 1×PCR Buffer with 1.5 mM of MgCl₂(Applied Biosystems), 200 μM of each dNTP, 1600 nM of each primer TK2S(5′-CCA TAG CAA CCG ACG TAC G-3′; SEQ ID NO: 24) and TKAS (5′-GAA TCGCGG CCA GCA TAG C-3′; SEQ ID NO: 25), The PCR cycling profile consistedof a first denaturation step 15 min at 94° C. followed by 38 cycles withdenaturation for 1 min of 95° C., annealing for 30 s at 65° C. andextension for 1 min at 72° C., and one final extension step of 10 min at72° C. Ten microliters of amplified product were analyzed by agarose gelelectrophoresis.

On the same samples PCR was performed in a 25 μl reaction mixturecontaining 100-500 ng of genomic DNA, 1×PCR Buffer with 1.5 mM of MgCl₂(Applied Biosystems), 200 μM of each dNTP, 300 nM of each primer HTK4+(5′-TTC TCT AGG CGC CGG AAT TCG TT-3′; SEQ ID NO: 20) and HTK2-(5′-ATCCAG GAT AAA GAC GTG CAT GG-3′; SEQ ID NO: 21), 1.25 U of AmpliTaq Gold(Applied Biosystems). The PCR cycling profile consisted of a firstdenaturation step 15 min at 94° C. followed by 40 cycles withdenaturation for 30 s at 95° C., annealing for 50 s at 600 C andextension for 1 min at 72° C., and one final extension step of 10 min at72° C. Ten microliters of amplified product were analyzed by agarose gelelectrophoresis.

cDNA Sequence Analysis of the TK3 Spliced Form

The lower band of the product amplified with HTK4+/HTK2-primers fromAm12/TK3#53 supernatant was sequenced by using an automated fluorescentDNA sequence apparatus. Sequence was carried out by PRIMM (Milan,Italy).

Results

HSV-tk RT-PCR from RNA of Culture Supernatants

Two PCR products were detected analyzing RNA from SFCMM-3 as well as TK3vectors, produced by Am12 and E86 packaging cell lines (FIG. 3). The TK3vector corresponds to the described scSFCMM-3 vector (Chalmers, 2001).The major product (upper band) corresponds to the unspliced form ofHSV-tk (1020 base pair (bp) with HTK4+/HTK2-primers; 401 bp withTKA1/TKB1 primers) in all the samples. The size of the minor product(lower band) in the SFCMM-3 samples corresponds to the described splicedform, in which 227 bp are deleted (Garin 2001, Blood 97:122-129).

On the contrary, the size of the minor product in the TK3 samples isslightly larger, suggesting the presence of a different spliced form, inwhich a smaller fragment is deleted; this difference between SFCMM-3 andTK3 samples is particularly evident with TK2S/TKAS primers. Additional,intermediate products observed with HTK4+/HTK2-primers only areconsidered as possible PCR artifacts.

HSV-tk PCR on DNA from Transduced Lymphocytes

Two PCR products were detected analyzing DNA from SFCMM-3 as well as TK3transduced PBLs (FIG. 4). The major product (upper band) corresponds tothe unspliced form of HSV-tk (644 bp with TK2S/TKAS primers; 1020 hpwith HTK4+/HTK2-primers). The size of the lower band (corresponding tothe described spliced form) in the SFCMM-3 samples is 417 bp withTK2S/TKAS printers and 793 bp with HTK4+/HTK2-primers.

Sequence of the Critical Portion of the HSV-tk Gene in SFCMM-3 VersusTK3.

The sequence of the critical portion of the HSV-tk gene in SFCMM-3versus TK3 is shown in FIG. 5. The gene segments which are deleted inthe spliced forms of SFCMM-3 and TK3 are indicated in italics. Thecorresponding splicing donor and acceptor sites are underlined.

The sequence data confirm that different spliced forms are generated inSFCMM-3 (227 bp deletion) and in TK3 (214 bp deletion) samples. Asexpected in both cases the GU and AG dinucleotides are present at 5′ and3′ borders of the deleted sequence, respectively.

EXAMPLE 3—ELIMINATION OF SPLICING BY SITE DIRECT MUTAGENESIS

Materials and Methods

Starting from the wild type SFCMM-3 vector, recombinant plasmids wereprepared by site-directed mutagenesis with different mutations in theHSV-tk gene (FIG. 6). Third-base degenerate changes were introduced thusin all instances the wild type amino acid sequence of HSV-tk enzyme waspreserved.

Site-Directed Mutagenesis

To generate pcDNA3.1-tk plasmid, SFCMM-3 plasmid was digested with EcoRIand XhoI and the EcoRI/XhoI fragment was ligated to EcoRI/XhoI digestedpoDNA3.1 (Invitrogen).

From the pcDNA3.1-tk plasmid, all the HSV-tk mutants were generated bysite-directed mutagenesis using the QuickChange Site-DirectedMutagenesis kit (Stratagene). The oligonucleotide primers used tointroduce the desired mutation were each complementary to oppositestrands of the vector. The sequences of sense primers are reported inthe following table:

Position SEQ of ID Mutant Plasmid mutation NO: Oligo sequence^(a)generated clone^(b) t330c 30 5′-CCTCGACCAGGG C GAGATATCGGCCG-3′ TkMut210.1 t330c 31 5′-CCTCGACCAGGG C GAGATATCGGCCG-3′ TkMut24 177.3 g555a 325′-ATGACCCCCCA A GCCGTGCT GGCG-3′ TkMut24 177.3 a541t/ 335′-TACCTTATGGGC TC CATGACCCCCCA A GCCGTGC-3′ TkMut34 57.1 g542c/ g555ag555a 34 5′-ATGACCCCCCA A GCCGTGCTGGCG-3′ TkMut4 19.1 t330c 355′-CCTCGACCAGGG C GAGATATCGGCCG-3′ TkMut223 171.1 a541t/ 365′-TACCTTATGGGC TC CATGACCCCCCA A GCCGTGC-3′ TkMut223 171.1 g542c/ g555a^(a)The position of nucleotide mutations are underlined in bold. ^(b)Thenumbers refer to the number of each SFCMM-3 HSV-tk plasmid clone mutant.

The resulting vectors were sequenced to confirm the corrected nucleotidesubstitutions. HSV-tk mutated fragment was then removed from pcDNA3.1 bydigestion with EcoRI and XhoI, then cloned into SFCMM-3 plasmid togenerate SFCMM-3 HSV-tk mutants.

After sequence confirmation of SFCMM-3 HSV-tk mutants (FIGS. 1A-1C), oneplasmid clone of each type was used to transiently transfect E86 cells.SFCMM-3 and TK3 plasmids were used as controls. The supernatant wascollected from the E86 cultures and used to stably transduce theamphotropic packaging cell line Am12 as described in Materials andMethods section of Example 2. The resulting cell populations wereselected for ΔLNGFR expression.

RNA was extracted from the E86 as well as Am12 culture supernatantscontaining infectious retroviral particles. RT-PCR reactions werecarried out on RNA from E86 and Am12 supernatants using TKA1 and TKB1primers according to the protocol described in Materials and Methodssection of Example 2.

Transduction and PCR Analysis of GEM A301 Cell Line and PBLs

CEM A301 cells were cultured in RPMI 1640 (Hyclone) supplemented with10% FBS (Hyclone) and 2 mM glutamine. CEM A301 cells were infected withAm12 culture supernatant containing SFCMM-3 HSV-tk mutants 2 and 234.The day before transduction 5×10⁵ cells/ml were cultured. Transductionwas then performed by one centrifugation cycle of retroviral vectorparticles on cells. After the transduction phase, cells were collectedand the percentage of actually transduced cells evaluated by FACSanalysis. PBLs from three different donors were transduced (as describedin Materials and Methods section of Example 2) with Am12 supernatantcontaining SFCMM-3 HSV-tk mutant 2 as well as SFCMM-3.

The transduced cells were then sorted for ΔLNGFR expression and expandeda few days in culture.

One pellet for PCR analysis was prepared from the transduced nonselected cells as well as from transduced selected cells as described inMaterials and Methods section of Example 2. Genomic DNA was extractedfrom pellet cells and PCR reaction was carried out with TKA1 and TKB1 orTKY1 and TKY2 or TKZ1 and TKZ2 primers (as indicated in the table below)in a 25 μl reaction mixture containing 100-500 ng of genomic DNA, 1×PCRBuffer with 1.5 mM of MgCl₂ (Applied Biosystems), 200 nM of each dNTPs,300 nM of each primer, 1.25 Units of AmpliTaq Gold (Applied Biosystems).The PCR cycling profile consisted of a first denaturation step for 15min at 94° C. followed by 40 cycles with denaturation for 30 s at 94°C., annealing for 50 s at 60° C. and extension for 1 min at 72° C., andone final extension step of 10 min at 72° C., and one final extensionstep of 10 min at 72° C. Ten microliters of amplified product wereanalyzed by agarose gel electrophoresis.

PCR product SEQ length Primer ID (HSV-tk name Primer sequence NO:unspliced) TKA1 5′-CGT ACC CGA GCC 22 401 bp GAT GAC TT-3′ TKB1 5′-TGTGTC TGT CCT 23 CCG GAA GG-3′ TKY1 5′-TTA TAT AGA CGG 26 542 bp TCC TCACGG G-3′ TKY2 5′-CCA GCA TAG CCA 27 GGT CAA GC-3′ TKZ1 5′-GCC ACC ATGGCT 28 1149 bp TCG TAC-3′ TKZ2 5′-CGA GTT AAT TCT 29 CAG TTA GCC TCC-3′ResultsSplicing Properties of HSV-tk Mutants in E86 Supernatants

The major product (upper band) corresponding to the unspliced form ofHSV-tk (401 bp) is detected in all the samples except the negativecontrol (supernatant from non transfected E86 cells, E86 C-) (FIG. 7).

The minor product (lower band) is less abundant in RNA from all themutant vectors, in respect to SFCMM-3 and TK3 vectors, produced by E86packaging cell lines.

Splicing Properties of HSV-tk Mutants in Am12 Supernatants

The unspliced form of HSV-tk is detected in all the samples except thenegative controls (supernatant from non infected cells, Am12 C-, andH₂O) (FIG. 8). A lower band is detected in TkMut4 (mutation 4) andTkMut34 (mutations 3+4) mutants, corresponding to the lower band of TK3and SFCMM-3 sample, respectively. The intensity of ethidium bromidesignal is lower compared to TK3 and SFCMM-3, indicating that the splicedproduct is less represented in the mutants. This hand is not detected inTkMut2 (mutation 2), TkMut234 (mutations 2+3+4) and TkMut24 (mutations2+4) mutants. Indeed, two very weak bands of approximately 100 and 200bp are detected in TkMut2, TkMut234 and TkMut24 mutants.

Splicing Properties of HSV-tk Mutants in Transduced CEM A301 Cells andPBLs

Genomic DNA from CEM A301 transduced with TkMut2 and TkMut234 mutants aswell as with SFCMM-3 and TK3 supernatants was analyzed by PCR usingTKA1/TKB1 primers. The unspliced form of HSV-tk (401 bp) is detected inall the samples except the negative controls (non transduced cells, CEMNT, and H₂O) (FIG. 9). A lower band is detected in TK3 and SFCMM-3preselection and postselection sample, corresponding to the spliced formof HSV-tk gene. No lower band is observed in CEM transduced with TkMut2and TkMut234 mutants, before and after selection.

To exclude the possibility of alternative splicing events occurring indifferent regions of HSV-tk gene, a more extensive analysis of CEM A301transduced with TkMut2 as well as with SFCMM-3 supernatants was doneusing TKY1/TKY2 and TKZ1/TKZ2 primers.

The unspliced form of HSV-tk (542 bp and 1149 bp, respectively) isdetected in all the samples (FIG. 10). A lower band is detected inSFCMM-3 preselection and postselection sample, corresponding to thespliced form of HSV-tk gene. No lower band is observed in CEM transducedwith TkMut2, nor with TKY1/TKY2 neither with TKZ1/TKZ2 PCR primers.

Genomic DNA from PBLs of three different donors (Don1, Don2, Don3)transduced with SFCMM-3 and/or TkMut2 supernatants was analyzed by PCRusing TKA1/TKB1 primers. The =spliced form of HSV-tk (401 bp) isdetected in all the samples (FIG. 11). A lower band is detected inSFCMM-3 sample, corresponding to the spliced form of HSV-tk gene. Nolower band is observed in PBLs transduced with TkMut2. The same resultwas obtained using TKZ1/TKZ2 primers (FIG. 12), which encompasses thefull-length HSV-tk gene (1149 bp), thus excluding the possibility thatthe introduced mutation could generated different splicing variants.

Overall these findings indicate that the introduced mutations abolish orat least very significantly decrease any HSV-tk gene spliced form intransduced CEM as well as in transduced lymphocytes.

TABLE 1 Analysis of HSV-tk spliced form on SFCMM-3 supernatants andtransduced PBLs % Spliced HSV-tk/Unspliced HSV-tk + SFCMM-3 vectorSpliced HSV-tk supernatant RNA DNA Lot number (supernatant) (transducedPBLs) 50.302-22 0.65 0.84 1.25 50.302-23 0.65 1.50 50.302-24 0.98 1.5850.302-26 0.72 1.00 0.92 2.02 02/047 1.10 3.86 03/087 2.60 4.00 mean1.12 1.89 sd 0.75 1.22 n 6 9 min 0.65 0.84 max 2.60 4.00

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

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The invention claimed is:
 1. A method of destroying cells comprising (i)introducing into said cells a polynucleotide selected from the groupconsisting of the TkMut23 polynucleotide as set forth in SEQ ID NO:3,the TkMut24 polynucleotide as set forth in SEQ ID NO:4, and the TkMut234polynucleotide as set forth in SEQ ID NO:7, and expressing the thymidinekinase encoded by the polynucleotide in said cells; and (ii)simultaneously, separately or sequentially contacting said cells with anon-toxic agent which is converted by the thymidine kinase to a toxicagent, thereby destroying the cells.
 2. The method according to claim 1,wherein the non-toxic agent is selected from the group consisting ofganciclovir, acyclovir, triflurothymidine, 1-[2-deoxy, 2-fluoro,β-D-arabino furanosyl]-5-iodouracil, ara-A, ara 1, 1-β-D arabinofuranosyl thymine, 5-ethyl-2′deoxyuridine,5-iodo-5′-amino-2,5′-dideoxyuridine, idoxuridine, AZT, AIV,dideoxycytidine, Ara C, and bromovinyl deoxyuridine (BVDU).
 3. A methodof treating a patient with cells in need of destruction comprising (i)introducing into the patient a polynucleotide selected from the groupconsisting of the TkMut23 polynucleotide as set forth in SEQ ID NO:3,the TkMut24 polynucleotide as set forth in SEQ ID NO:4, and the TkMut234polynucleotide as set forth in SEQ ID NO:7, and expressing the thymidinekinase encoded by the polynucleotide in said cells; and (ii)simultaneously, separately or sequentially introducing into the patienta non-toxic agent which is converted by the thymidine kinase to a toxicagent, thereby destroying the cells and treating said patient.
 4. Themethod according to claim 3, wherein the non-toxic agent is selectedfrom the group consisting of ganciclovir, acyclovir, triflurothymidine,1-[2-deoxy, 2-fluoro, β-D-arabino furanosyl]-5-iodouracil, ara-A, ara 1,1-β-D arabino furanosyl thymine, 5-ethyl-2′deoxyuridine,5-iodo-5′-amino-2,5′-dideoxyuridine, idoxuridine, AZT, AIV,dideoxycytidine, Ara C, and bromovinyl deoxyuridine (BVDU).
 5. A methodof treating a patient with cells in need of destruction comprising (i)removing the cells from the patient or donor cells; (ii) introducinginto the cells ex vivo a polynucleotide selected from the groupconsisting of the TkMut23 polynucleotide as set forth in SEQ ID NO:3,the TkMut24 polynucleotide as set forth in SEQ ID NO:4, and the TkMut234polynucleotide as set forth in SEQ ID NO:7; (iii) introducing themodified cells into the patient; (iv) allowing the cells to express thethymidine kinase encoded by the polynucleotide; and (v) administering tothe patient a non-toxic agent which is converted by the thymidine kinaseinto a toxic agent, thereby destroying the cells and treating saidpatient.
 6. The method according to claim 5, wherein the non-toxic agentis selected from the group consisting of ganciclovir, acyclovir,triflurothymidine, 1-[2-deoxy, 2-fluoro, β-D-arabinofuranosyl]-5-iodouracil, ara-A, ara 1, 1-β-D arabino furanosyl thymine,5-ethyl-2′deoxyuridine, 5-iodo-5′-amino-2,5′-dideoxyuridine,idoxuridine, AZT, AIV, dideoxycytidine, Ara C, and bromovinyldeoxyuridine (BVDU).
 7. A method of treating graft-versus-host diseasein a patient comprising: (i) administering to the patient T-cellsgenetically engineered to include a polynucleotide selected from thegroup consisting of the TkMut23 polynucleotide as set forth in SEQ IDNO:3, the TkMut24 polynucleotide as set forth in SEQ ID NO:4, and theTkMut234 polynucleotide as set forth in SEQ ID NO:7, and expressing thethymidine kinase encoded by the polynucleotide in said T-cells; and (ii)administering to said patient, on occurrence of graft-versus-hostdisease, a non-toxic agent which is converted by the thymidine kinase toa toxic agent thereby killing said genetically engineered T-cells andtreating the graft-versus-host disease in said patient.
 8. The methodaccording to claim 7, wherein the non-toxic agent is selected from thegroup consisting of ganciclovir, acyclovir, triflurothymidine,1-[2-deoxy, 2-fluoro, β-D-arabino furanosyl]-5-iodouracil, ara-A, ara 1,1-β-D arabino furanosyl thymine, 5-ethyl-2′deoxyuridine,5-iodo-5′-amino-2,5′-dideoxyuridine, idoxuridine, AZT, AIV,dideoxycytidine, Ara C, and bromovinyl deoxyuridine (BVDU).